Cholera, an acute diarrheal disease, remains a global burden to human health. The key factor chiefly responsible for this devastating disease is cholera toxin, an AB5 toxin that is produced and secreted by Vibrio cholerae. Its extracellular secretion is dependent on the type II secretion system (T2SS), which is also responsible for the outer membrane translocation of proteases, lipases, nucleases and chitinases. Common to many Gram-negative pathogens, it uniquely transports these factors from the periplasmic compartment to the extracellular environment in their fully folded conformations. Despite a dramatic increase in structural knowledge of the T2SS and its individual components in recent years, the mechanism by which T2S substrates are recognized and sorted for outer membrane translocation remains to be determined. A lack of significant sequence and structural similarity between these proteins complicates the identification of a common secretion signal. Other confounding factors include the findings that the T2SS supports the extracellular transport of both soluble proteins and lipoproteins and that there are differences in the final destination among T2S substrates. Some T2S substrates such as cholera toxin are released to the extracellular space once transported through the outer membrane; however, others remain surface associated or may reattach to the bacterial cell surface following extracellular release. The trypsin-like protease VesB is an example of a T2S substrate that is primarily retained on the cell surface. VesB belongs to a unique class of extracellular enzymes that have a C-terminal extension consisting of two prominent glycines and a hydrophobic helix followed by positively charged residues (GlyGly-CTERM domain). Rhombosortase, a newly discovered member of the intramembrane rhomboid protease family, cleaves off the GlyGly-CTERM domain during transit of VesB through the cell envelope, and the posttranslationally modified VesB is localized to the cell surface. The experiments described in this proposal are designed to test the hypothesis that the T2S system, in collaboration with rhombosortase, mediates the maturation and surface localization of GlyGly-CTERM containing proteins. Specifically, this proposal will determine the mechanism of surface anchoring of proteins produced with GlyGly-CTERM extensions, decipher how GlyGly-CTERM proteins are differentially recognized and secreted by the T2SS, and assess the T2SS/rhombosortase system for surface localization of GlyGly-CTERM-tagged heterologous proteins. The findings will facilitate understanding of the function and specificity of rhombosortase as well as the broader class of medically relevant rhomboid proteases and may identify ways to manipulate the T2S/rhombosortase system for preventative use.